Calcification of intervertebral discs in the dachshund: a radiographic ...

Acta Veterinaria Scandinavica
BioMed Central
Research
Calcification of intervertebral discs in the dachshund: a radiographic
and histopathologic study of 20 dogs
Øyvind Stigen* 1 and Øyvor Kolbjørnsen2 Open Access
Address: 1Department of Companion Animal Clinical Sciences, Norwegian School of Veterinary Science, PO Box 8146, 0033 Oslo, Norway and
2Department of Pathology, National Veterinary Institute, PO Box 8156, 0033 Oslo, Norway
Email: Øyvind Stigen* - oyvind.stigen@veths.no; Øyvor Kolbjørnsen - oyvor.kolbjornsen@vetinst.no
* Corresponding author
Published: 21 December 2007
Acta Veterinaria Scandinavica 2007, 49:39 doi:10.1186/1751-0147-49-39
This article is available from: http://www.actavetscand.com/content/49/1/39
Abstract
Background: The purpose of the study was to compare radiographic and histopathologic findings
with regard to number and extent of calcified discs in the dachshund.
Methods: The intervertebral discs of 20 dachshunds were subjected to a radiographic and
histopathologic examination. The dogs were selected randomly from clinical cases euthanased for
reasons unrelated to research at the Norwegian School of Veterinary Science. Lateral radiographs
were taken of the vertebral columns after removing them from the carcasses. The histopathologic
examination included 5 µm thick sections in the transverse plane, stained with hematoxylin-eosin
and von Kossa. Radiographs and histological sections were evaluated independently.
Results: A total of 148 (28.5%) calcified discs were identified at the radiographic and 230 (45.7%)
at the histopathologic examination. Of 92 discs found to be calcified by histopathology, but not by
radiography, the degree of calcification was evaluated as 'slight' in 84 (91.3%). All the intervertebral
discs (n = 138) that were found to be calcified by radiography were also found to be calcified by
histopathology.
Conclusion: A sensitivity of 0.6 and specificity of 1.0 for radiography was calculated when using
histopathology as the gold standard.
Background
Intervertebral disc disease (IDD) is the most common
cause of neurologic dysfunction in the dog [1]. The disease
is most often diagnosed in dachshunds, and in 8117
canine cases of IDD, Priester [2] found 3898 (48.0%) to
occur in this breed. The breed prevalence of IDD in the
dachshund has been estimated to be 19.0% [3].
In dachshunds and other chondrodystrophoid breeds,
IDD is mainly due to extrusion of nuclear material from
the disc into the vertebral canal. Hansen [4] classified this
Received: 26 April 2007
Accepted: 21 December 2007
© 2007 Stigen and Kolbjørnsen; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
type of disc disease as protrusion of type 1 and found
chondroid degenerative changes of the disc with subsequent
dystrophic calcification of the nucleus to be significant
preceding factors. In accordance with this,
radiographic studies have shown that young dachshunds
with calcified intervertebral discs are predisposed to IDD
[5,6].
The occurrence of calcified discs is found to differ between
dachshunds of the same age, size and coat variety [7]. Furthermore,
a genetic factor is shown to be essential for the
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occurrence of calcified discs in a dog and the heritability
in Norwegian dachshunds is estimated to be 0.15 and
0.22 [8]. In Danish wirehaired dachshunds Jensen and
Christensen [9] estimated the heritability to be 0.60 and
0.87. Thus, the potential exists for a breeding programme
that could reduce the occurrence of calcified discs and
thereby IDD in this breed.
Low-field magnetic resonance (MR) imaging is found to
be very sensitive in the evaluation of disc degeneration in
dogs [10]. However, the cost of MR imaging is high and
the number of MR machines for veterinary usage is limited.
For screening purposes, a radiographic examination
is therefore still the most suitable method for the identification
of calcified discs in a large number of live dachshunds.
Information obtained by reading radiographs can
be included as part of a breeding programme.
Different degrees of calcification are found in degenerated
chondrodystrophoid discs [11,12]. Discs with minor calcifications
could be difficult to identify on radiographs.
Also, non-calcified discs could be misjudged and read as
calcified. By this, false-positive and false-negative errors
may be made in reading radiographs for calcified discs. To
the authors' knowledge the extent of such errors has not
been reported. However, in a pathologic study of 16 oneyear
old chondrodystrophoid dogs Hansen [4] found calcified
discs in at least 10 (62.5%) of the dogs, while in a
radiographic study of 327 one-year old dachshunds Stigen
[7] found calcified discs in 79 (24.2%) of the dogs. On the
Table 1: Case material.
assumption that most of the dogs in the former study were
dachshunds, these two studies together indicate that a
radiographic study is far less sensitive than a pathologic
study in identification of calcified discs. The present study
was performed to test this hypothesis.
Methods
The vertebral columns were obtained from 20 dachshunds
that were euthanased for reasons unrelated to
research at the Department of Companion Animal Clinical
Sciences, Norwegian School of Veterinary Science. The
case material, including size and coat varieties, sex, age
and reason for euthanasia is presented in Table 1. The age
of the dachshunds ranged from ten months to 13 years
(mean 5.3 years) and there were twelve (60.0%) females.
The vertebral columns were separated from the skull, ribs
and pelvis by dearticulation. The tail, including the coccygeal
vertebrae, was also removed. The spines were freed of
muscle, loose connective tissue and ligaments.
Within 24 hours of death, conventional lateral radiographs
were taken of the cervical, thoracic, lumbar and
sacral vertebral columns [13]. The radiographic equipment
was a Philips Medio 50 CP generator and a Super
Rotalix 2550 tube. The cassettes were Cawo 18 × 24 cm
rectangle x-ray cassettes containing a green-emitting 100
MR (fine) intensifying screen on one side of a Kodak T-
Mat L/RA-film. No grid was used and at exposure the specimens
were in direct contact with the cassettes. The voltage
Dog no. Variety Sex Age, years Reason for euthanasia
Size, Coat
1 Dwarf, Smoothcoated � 8 Neurological disorder
2 Dwarf, Longhaired �

Acta Veterinaria Scandinavica 2007, 49:39 http://www.actavetscand.com/content/49/1/39
and milliampere-seconds used depended upon the size of
the vertebrae and varied between 50 and 60 kV and 10
and 20 mAs respectively.
At least four exposures were taken of each dog covering
the vertebral column from the first cervical (C1) to the
first sacral (S1) vertebra. The total number of calcified
discs and their location in the vertebral column were
recorded according to earlier described methods [13,14].
Three degrees of calcification of the individual discs were
noted and defined as follows (Figure 1):
SLIGHT:
- one or more calcified bodies with diameter less than 2
mm, or
- calcification of part of the transitional zone between the
nucleus pulposus and the annulus fibrosus, or
- indistinct calcification of a larger part of the nucleus pulposus
MODERATE:
- calcified bodies with diameter less than 2 mm and calcification
of part of the transitional zone between the
nucleus pulposus and the annulus fibrosus, or
- distinct calcification of a larger part of the nucleus pulposus,
or
- indistinct calcification of the entire nucleus pulposus
Lateral thoracic dachshund Figure radiograph 1(T5)
of vertebra standard from in size the a six-year-old, (Dog seventh no. cervical 10) male, (C7) smoothcoated to the fifth
Lateral radiograph from the seventh cervical (C7) to the fifth
thoracic (T5) vertebra in a six-year-old, male, smoothcoated
dachshund of standard size (Dog no. 10). Three intervertebral
discs; T1–2, T3–4 and T4–5, shows a 'severe', 'moderate'
or 'slight' degree of calcification (arrows).
SEVERE:
- distinct calcification of the entire nucleus pulposus
If there was doubt about calcification or the degree of calcification,
additional radiographs were taken of the vertebral
section of current interest.
Immediately after the radiographic examination, specimens
for histological examination were collected by
removing each intervertebral disc between the second cervical
(C2) and first sacral (S1) vertebra in all of the 20 vertebral
columns. With tissue forceps, a scalpel handle and
no. 11 blade the discs were carefully separated from the
adjacent vertebral body end plates (extremitas cranialis et
caudalis) and removed.
All specimens (520 discs) were fixed in 4% phosphatebuffered
formaldehyde, pH 7.2. Subsequently the discs
were dehydrated in ethanol, equilibrated in xylene and
embedded in paraffin. Decalcifications of the specimens
were not performed. The discs were sectioned transversely
at about 5 µm and stained with hematoxilin-eosin and
von Kossa, a method for demonstration of calcium [15].
During the preparation process all sections concerning 17
discs became incomplete and consequently these discs
were regarded as withdrawals. By this, 503 discs were
available for a final histopathologic examination by light
microscopy.
Three degrees of calcification of the individual discs were
noted, and for each category, all findings might appear
either separately or in combination with other lesions.
The degrees of calcification were defined as follows:
SLIGHT (Figure 2):
- single or multiple foci of calcification occupying less
than half of the area of the nucleus pulposus
- single or multiple small foci of calcification within the
annulus fibrosus
MODERATE (Figure 3):
- single or multiple foci of calcification occupying more
than half, but not all, of the area of the nucleus pulposus
- single or multiple medium size foci of calcification
within the annulus fibrosus
- thin calcified ring, continuous or discontinuous, either
surrounding the nucleus pulposus or localised within the
annulus fibrosus
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Histopathology Figure 2 of disc no. 6 (C7–T1) from Dog no. 10
Histopathology of disc no. 6 (C7–T1) from Dog no. 10. Calcium
deposits (arrows) are seen multifocally occupying less
than half of the area of the nucleus pulposus (Np): 'slight'
degree of calcification (1). Af = annulus fibrosus. Von Kossa;
Bar = 1.6 mm.
SEVERE (Figure 4):
- widespread or total calcification of the nucleus pulposus
- single or multiple large foci of calcification within the
annulus fibrosus
- broad calcified ring, continuous or discontinuous, either
surrounding the nucleus pulposus or localised within the
annulus fibrosus
If two or more sections of the same disc were read to have
different degrees of calcification, the section with the most
severe degree of calcification determined the evaluation of
that disc.
Radiographs and histological sections were evaluated
independently.
Results
At the radiographic examination, calcification was found
in 148 (28.5%) of the 520 discs. Of all affected discs, a
severe degree of calcification was found in 15, a moderate
degree in 43 and a slight degree in 90 discs. Calcified discs
were identified in 18 (90.0%) of the dogs and the number
of calcified discs in each affected dog ranged from one
(dog no. 19) to 20 (dog no. 15).
Histopathology of disc no 10 (T4–5) from Dog no. 10
Figure 3
Histopathology of disc no 10 (T4–5) from Dog no. 10. Calcium
deposits (arrows) form a thin discontinuous ring surrounding
the nucleus pulposus (Np), with one focus
extending into the annulus fibrosus: 'moderate' degree of calcification
(2). Af = annulus fibrosus. Von Kossa; Bar = 1.6
mm.
At the histopathologic examination, calcification was
found in 230 (45.7%) of the 503 discs. Of all affected
discs, a severe degree of calcification was found in 30, a
moderate degree in 45 and a slight degree in 155 discs.
Calcified discs were identified in all the dogs and the
number of calcified discs in each dog ranged from one
(dog no. 19) to 24 (dog no. 17).
The individual number and location of calcified discs
together with the degree of calcification of each affected
disc, identified both by radiography and histopathology,
are presented in Additional file 1, Table 3.
Comparative results by radiographic and histopathologic
examination concerning 503 discs are presented in Table
2. Of all the discs found to be calcified by histopathology
(n = 230), 138 (60.0%) were found to be calcified by radiography.
All the discs found to be calcified by radiography
(n = 138) were also found to be calcified by histopathology.
Using histopathology as the gold standard, a sensitiv-
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Histopathology Figure 4 of disc no. 7 (T1–2) from Dog no. 10
Histopathology of disc no. 7 (T1–2) from Dog no. 10. Calcium
deposits (arrows) in the nucleus pulposus (Np) and in a
broad discontinuous ring within the annulus fibrosus (Af):
'severe' degree of calcification (3). Von Kossa; Bar = 1.6 mm.
ity of 0.6 (138/230) and a specificity of 1.0 (273/273) was
calculated for the radiographic examination.
In 95 discs the degree of calcification was found to be
equal in histopathology and radiography. In 125 discs the
degree of calcification was higher in histopathology and
in 10 it was higher in radiography.
Discussion
The present study shows that a significantly higher
number (45.7% versus 27.4%) of calcified discs are identified
by a histopathologic examination compared to a
radiographic examination of intervertebral discs in dachshunds.
This finding verifies the hypothesis presented at
the beginning of the present paper.
Table 2: The distribution of 503 intervertebral discs on the basis
of calcification (+) or not (-) evaluated by a radiographic and a
histopathologic examination.
Histolopathology Total
+ -
Radiology + 138 (27.4%) 0 138 (27.4%)
- 92 (18.3%) 273 (54.3%) 365 (72.6%)
Total 230 (45.7%) 273 (54.3%) 503 (100.0%)
At the radiographic examination, a total of 148 calcified
discs were identified in 18 dogs. This makes a mean of 8.2
calcified discs in each affected individual. Both the percentage
number of dogs with calcified discs (90.0%) and
the mean number of such discs in each affected dog are
higher than values for corresponding parameters found in
previous radiographic studies [6,7,12]. The fact that the
present study is related to clinical cases, whereas the previous
radiographic studies were related to clinically normal
dachshunds, could be the reason for this disparity.
In dogs no. 2 and 3, no calcified discs were found at the
radiographic examination, but at the histopathologic
examination a slight degree of calcification was found in
five and 15 discs, respectively. This finding shows that a
total absence of calcified discs visible on radiographs of a
dachshund is no guarantee for the dog not having extensive
calcification at histopathologic examination.
Of the 92 discs that were found to be calcified only at the
histopathologic examination, 84 (91.3%) were noted to
have a 'slight' degree, eight a 'moderate' degree and none
a 'severe' degree of calcification. This agrees with the general
understanding that an ordinary radiographic examination
of the vertebral column in dogs is not very sensitive
at detecting minor calcifications of intervertebral discs.
The radiographic examination in the present study was
done on vertebral columns separated from the skull, ribs
and pelvis and freed of soft tissue. This meant that superimposition
of tissue adjacent to the discs (e.g. caput costae
and os ilium) was avoided on the radiographs. Also
motion blurring was not a problem with the examination
of dead specimens. These factors contributed to high technical
quality of the radiographs. Therefore, the sensitivity
of the radiographic examination in the present study is
likely higher than could be expected in similar studies on
live dogs. As a consequence, the presented results concerning
total number of calcified discs and degree of calcification
of individual discs, cannot directly be compared to
results from previous radiographic studies [5-7,9,12] on
live dogs.
The 20 dachshunds included in the present study were
euthanased up to 24 hours before being available for the
radiographic examination. As a result, rigor mortis was
present in several dogs and for these the standard procedure
for spinal radiography of live dogs [13,16] was inappropriate.
Two dogs (no. 3 and 18) had been subjected to
post-mortem examinations immediately after euthanasia
and their vertebral columns were thereafter included in
the study. To achieve radiographs of high technical quality
from more dogs, separated vertebral columns, instead of
vertebral columns "in situ", were chosen for radiography.
Diagnostic imaging of separated vertebral columns is pre-
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viously reported in radiographic [17,18] and MR [10]
examinations in dogs.
Radiographs and histological sections were each evaluated
by one person, which means that both the radiographic
and the histopathologic examination were subject
to significant interobserver variation. With two or more
persons evaluating the radiographs and the histological
sections respectively, the interobserver variation and consequently
the standard deviations of the observations presented
in Table 2, would decrease. Consequently, the
calculated sensitivity of 0.6 represents an underestimate.
At the radiographic examination, additional radiographs
were taken if there was any doubt about calcification. At
the histopathologic examination, at least two complete
histological sections of each disc were examined to
remove any doubt about calcification. By this, the present
study should not be encumbered with significant intraobserver
variation.
A specificity of 1.0 indicates that no false-positive errors
were made at the radiographic examination. The high
technical quality of the radiographs is probably a significant
reason for this. Another reason could be a reserved
attitude by the reader of the radiographs, evaluating discs
with uncertain calcification as negative.
Von Kossa, a special stain for identification of calcium,
was included in the histopathologic examination of all
intervertebral discs. In previous histopathologic examinations
of disc degeneration in dogs [4,10], this stain was
not used. In the study by Seiler et al. [10], decalcification
was also part of the preparation for histopathologic examination.
A discrepancy in results can easily arise when different
studies adopt different protocols.
In the present study, histopathology was found considerably
more sensitive than radiography in identifying calcified
discs. Nevertheless, in ten discs the degree of
calcification was found to be higher in radiography than
in histopathology. A histopathologic examination of non
representative sections could be an explanation for the
different findings in these ten discs.
Conclusion
A histopathologic examination is superior to a radiographic
examination in identification of calcified intervertebral
discs in the dachshund. A sensitivity of 0.6 and a
specificity of 1.0 for radiography is calculated using
number of calcified discs as parameter and histopathology
as the gold standard. Calcification of the nucleus pulposus
and/or the annulus fibrosus is a significant sign of
disc degeneration in dachshunds and the distribution of
detectable calcification in affected discs represents the distribution
of a continuous variable.
Competing interests
The author(s) declare that they have no competing interests.
Authors' contributions
ØS conceived of the study, carried out the radiographic
examination and is the main author of the paper. ØK carried
out the histopathologic examination. Both authors
read and approved the final manuscript.
Additional material
Additional file 1
Table 3. Degree of calcification, evaluated by a radiographic and a histopathologic
examination in 26 intervertebral discs from each of 20 dachshunds.
Degrees of calcification evaluated by radiography: light grey:
slight. dark grey: moderate. black: severe. Degrees of calcification evaluated
by histopathology: 1 slight. 2 moderate. 3 severe. Other note: -:
intervertebral disc not available for a final histopathologic examination.
Click here for file
[http://www.biomedcentral.com/content/supplementary/1751-
0147-49-39-S1.doc]
Acknowledgements
We are grateful to Ms. Birgit Røe and Ms. Anne-Kristine Fevang for performing
the histopathologic sections, to Dr. Agnar Kvellestad for practical
help with photographic editing and to Dr. Monica Theresa Johnsen for language
consultation.
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